1. The Field of the Invention
The present invention relates to methods and apparatus for the isoelectric focusing of amphoteric substances. More particularly, the present invention increases the resolution and separation characteristics of amphoteric biological substances by utilizing a barrier between the electrode compartments and the focusing cell compartments which has the properties of a bipolar selective membrane.
2. The Background of the Invention
Numerous areas of modern biology and genetic engineering depend on the availability of large quantities of high purity proteins. Currently available methods of protein purification include many kinds of chromatographic and electrophoretic techniques. Among these techniques, isoelectric focusing (hereinafter "IEF") has many attractive features.
The principle of IEF is based on the fact that certain biological materials (such as proteins, peptides, nucleic acids, and viruses) and even some living cells are amphoteric in nature--i.e., they are positively charged in an acidic media and negatively charged in a basic media. At a particular pH value, called the isoelectric point (hereinafter "pI"), these biomaterials will have a zero net charge.
Being charged in a pH gradient, the biomaterials migrate under the influence of an electric field until they reach the pH of their isoelectric point. At the isoelectric point, by virtue of their zero net charge, the biomaterials are not influenced by the electric field. Diffusion of "focused" biomaterials away from their pI will cause them to once again become charged, whereby they will electrophoretically migrate back to their pI. Thus, the biomaterials focus into narrow zones (defined by the pH of the medium and the electric field applied) from which the biomaterials can be selectively separated.
In one known method of isoelectric focusing, the pH gradient is established by the introduction of carrier ampholytes into the electric field. "Carrier ampholytes" are defined as ampholytes of relatively low molecular weight having conductance as well as buffer capacity, in the isoelectric state. Mixtures of synthetic polyaminopolycarboxylic acids have been used as carrier ampholytes.
In order to establish suitable pH gradients for IEF, it is necessary to have access to a great number of carrier ampholytes with isoelectric points well distributed along the pH scale. A commercial mixture of such amphoteric substances (called "Ampholine") is available from LKB Produkter AB, a Swedish Company. Ampholine is thought to be principally composed of polyaminopolycarboxylic acid molecules made by reacting polyamines with acrylic acid.
By manipulating the pH range of the carrier ampholytes, isoelectric focusing has the potential for high resolving power However, the potential of isoelectric focusing as a means for separating amphoteric substances has not been realized because of the time necessary and the quality of separation of prior art processes.
Since acids are attracted to the anode of the electric field and bases to the cathode during electrolysis, an increasing pH gradient from the anode to the cathode will develop in a convection free electrolytic conductor. The success of isoelectric focusing depends on the satisfaction of three conditions: (1) that the pH gradient is stable in time; (2) that an electrolyte deficit does not develop within the field, thereby tending to quench the current and/or give rise to local overheating; and (3) that the pH gradient--d(pH)/dx--has a low value in the pH region of interest in the actual separation.
Isoelectric focusing is most often practiced in small-scale batch instruments where the fluid is stabilized by either gels or density gradients established by a non-migrating solute such as sucrose. The capacity of such instruments for product separation is generally limited by the cross-sectional area of the apparatus. Because the apparatus cross-section is limited by the need to dissipate the heat generated by the electric field, larger scale preparative work has been proposed using continuous flow and recycling techniques.
One known technique which comes close to combining high resolution with large quantitative capacity is the recycling isoelectric focusing method disclosed in U.S. Letters Pat. No. 4,204,929 and No. 4,362,612.
Currently known recycling isoelectric focusing (hereinafter "RIEF") techniques involve dividing a fluid containing carrier ampholytes into a plurality of reservoirs and passing the contents of the reservoirs through an isoelectric focusing cell. The isoelectric focusing cell separates the fluids from adjacent reservoirs with ion non-separates selective permeable membranes which allow interchange of fluid constituents from channel to channel, but which inhibit bulk fluid flow. Electrodes establish an electrical potential transverse to the fluid flow thereby creating a pH gradient between successive channels.
The fluid from each reservoir exiting the isoelectric focusing cell is pumped to the reservoir which feeds the isoelectric focusing cell. A heat exchanger cools the fluid within the reservoirs. As the fluid is pumped into the top of the reservoir, the fluid is directed from the bottom of the reservoir back into the isoelectric focusing cell.
One problem with this technique is the continual remixing of purified materials with semi-purified or crude starting materials. Another serious drawback with the current RIEF techniques is dissipating the joule heat generated during the isoeleotric focusing process. Solutions to these problems are disclosed in copending U.S. patent application Ser. No. 07/320,725 in the name of the inventors hereof which has been incorporated by reference.
The use of dilute acids and bases for electrolyte solutions in the electrode chambers has been standard practice for virtually all isoelectric focusing systems. Typically the solutions used are 0.1 M sodium hydroxide (pH 12.5) for the catholyte and 0.1 M phosphoric acid (pH 2.3) for the anolyte. As the ampholytes focus, the subsequent pH gradient tends to be between these two pH extremes. Even when attempting to create shallow gradients with narrow range ampholytes, the extreme ends adjacent to the electrolyte solutions buffer to the electrolyte pH. This phenomenon is known as "spiking" or "acid notching".
In many isoelectric focusing applications spiking is not a major concern. However, in applications having a small number of separation channels or other limited area of separation, it is important to reduce or eliminate spiking to achieve high resolution separations. Otherwise the spiking tends to inhibit the formation of a narrow pH gradient which is important in achieving high resolution.
From the foregoing, it will be appreciated that what is needed in the art are apparatus and methods for isoelectric focusing of amphoteric substances which inhibit spiking.
Additionally, it would be a significant advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which enable narrow pH gradients to be obtained and maintained without significant spiking in the separation channels adjacent the electrodes.
It would be another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which provide high resolution separations.
Such methods and apparatus are disclosed and claimed herein.